Evolutionarily conserved amino acids in MHC-II mediate bat influenza A virus entry into human cells

The viral hemagglutinins of conventional influenza A viruses (IAVs) bind to sialylated glycans on host cell surfaces for attachment and subsequent infection. In contrast, hemagglutinins of bat-derived IAVs target major histocompatibility complex class II (MHC-II) for cell entry. MHC-II proteins from various vertebrate species can facilitate infection with the bat IAV H18N11. Yet, it has been difficult to biochemically determine the H18:MHC-II binding. Here, we followed a different approach and generated MHC-II chimeras from the human leukocyte antigen DR (HLA-DR), which supports H18-mediated entry, and the nonclassical MHC-II molecule HLA-DM, which does not. In this context, viral entry was supported only by a chimera containing the HLA-DR α1, α2, and β1 domains. Subsequent modeling of the H18:HLA-DR interaction identified the α2 domain as central for this interaction. Further mutational analyses revealed highly conserved amino acids within loop 4 (N149) and β-sheet 6 (V190) of the α2 domain as critical for virus entry. This suggests that conserved residues in the α1, α2, and β1 domains of MHC-II mediate H18-binding and virus propagation. The conservation of MHC-II amino acids, which are critical for H18N11 binding, may explain the broad species specificity of this virus.


Introduction
Zoonotic transmission of viruses represents a constant threat to global health. Bats play an important role as reservoir hosts for diverse, potentially deadly viral pathogens [1][2][3][4]. However, until recently, bats were not recognized as a reservoir for influenza A viruses (IAVs); rather, all IAV strains were believed to have originated from wild waterfowls [5]. This notion PLOS Biology | https://doi.org/10.1371/journal.pbio.3002182 July 6, 2023 1 / 18 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 was challenged by the discovery of the genome sequences of 2 novel IAV strains, H17N10 and H18N11, in New World bats [6][7][8]. Although these bat IAVs essentially resemble conventional IAVs, their surface glycoproteins (H17/18 and N10/11) differ fundamentally in function from those of conventional IAVs despite structural similarity. While the hemagglutinins of conventional IAV (H1 to H16) mediate attachment and entry via binding to sialic acid residues, both H17 and H18 are unable to bind sialylated glycans [8][9][10] and instead utilize major histocompatibility complex class II (MHC-II) molecules for cell entry [11]. MHC-II molecules are heterodimeric transmembrane proteins essential for adaptive immune responses as they present antigenic peptides of extracellularly derived proteins on the surface of professional antigen presenting cells to CD4 + T cells [12][13][14]. They consist of alpha (α)-and beta (β)-chains made of membrane-proximal barrel-shaped, immunoglobulin (Ig)like α2 and β2 domains and juxtaposed membrane-distal domains (α1 and β1), which contribute almost equally to the formation of the peptide-binding groove [13,15,16]. MHC-II molecules fold in the endoplasmic reticulum, from which they are transported with the help of the invariant chain to late endosomal compartments, where the invariant chain is degraded and MHC-II loaded with peptides [13,14]. This peptide loading is facilitated by a chaperone, the nonclassical MHC-II molecule DM (in human HLA-DM) [14,17]. HLA-DM resides in the late endosomal compartments and shares high structural similarity with classical MHC-II molecules but does not have a functional peptide-binding groove [14,18]. After binding a peptide, MHC-II molecules are trafficked to the plasma membrane [19].
The H18 binding site on MHC-II is unknown. However, classical MHC-II molecules from all vertebrate species tested to date enable H18-mediated infection [11], suggesting that highly conserved MHC-II residues facilitate viral entry. Here, we sought to define these residues through a mutational approach in which we generated chimeric MHC-II molecules from permissive classical HLA-DR and nonpermissive nonclassical HLA-DM molecules. We identified conserved residues within the α2 domain of HLA-DR as key for H18N11 infection.

The β2 domain of HLA-DR is dispensable for H18-mediated entry
To test whether the intracellular nonclassical MHC-II molecule HLA-DM can support H18-mediated cell entry, we generated a chimeric HLA-DM with its transmembrane and cytoplasmic domains swapped for those of HLA-DR (HLA-DMtc) (Figs 1A, S1A and S1B). Following transfection of human embryonic kidney (HEK) 293T cells with plasmids coding for HLA-DR, HLA-DM, and HLA-DMtc, we detected efficient surface expression of HLA-DMtc similar to that of HLA-DR by flow cytometry (Figs 1B and S2, S1 Data). However, only cells expressing HLA-DR but neither wild-type HLA-DM nor HLA-DMtc were able to support entry of a GFPencoding vesicular stomatitis virus whose glycoprotein was replaced by H18 (VSV-H18) [20] (Fig 1C), suggesting that the HLA-DM structure is incompatible with H18 binding.
We then generated further HLA-DR/DM chimeras by replacing individual domains of the α-and β-chains of HLA-DR with the corresponding domains of HLA-DM (Figs 1D, S1A and S1B). As shown in Fig 1E and S1 Data, all chimeras were expressed at the cell surface at levels comparable to HLA-DR except for chimeras 8 and 13, which showed lower cell surface expression. Irrespective of cell surface expression levels, only chimera 7, comprising the α1, α2, and β1 domains of HLA-DR in assembly with the β2 of HLA-DM, allowed infection with VSV-H18 (Figs 1F and S3A, S1 Data), suggesting that the β2 domain of HLA-DR is dispensable for virus entry. To test this, we coexpressed the full-length HLA-DR α-chain with truncated versions of the HLA-DR β-chain consisting of the β1 domain with short (8, 15, or 20 residues) fragments of the β2 domain retained at its C-terminus ( Fig 1G). These truncated  Table). The models revealed the α2 domain as essential for the interaction with H18, making up between 66.7% (26 of 39 residues in model 1) and 73.3% (22 of 30 residues in model 2) of the putative H18-binding site, with the α1 domain contributing between 16.7% (5 out of 30 residues, model 2) and 25.6% (10 of 39 residues, model 1) and the β1 domain only 3 residues to the interacting surface (Figs 2B and S4A and S1 Table). Accordingly, the α2 domain buried most interface in both models: 940 Å 2 (model 1) or 740 Å 2 (model 2), compared to 330 Å 2 (model 1) or 210 Å 2 (model 2) contributed by the α1 domain, and 100 Å 2 (model 1) or 170 Å 2 (model 2) by the β1 domain (Figs 2B and S4A and S1 Table).

PLOS BIOLOGY
Since the α2 domain makes up the majority of the putative H18 binding surface, we set out to identify the regions critical for viral cell entry within this domain. Based on chimera number 6 (α1, β1, β2 of HLA-DR, α2 of HLA-DM; Fig 1D), we generated MHC-II constructs with chimeric HLA-DR/DM α2 domains, replacing secondary structure motifs (loops and β-sheets) in the α2 domain of HLA-DR with their corresponding HLA-DM counterparts (Fig 2C and 2D). The loops 1 to 3 and β-sheets 1 to 2 form the core of the Ig-like fold and thus are not surface exposed in the HLA-DR crystal structure (S4B Fig). Therefore, we assumed that they should not be involved in H18-mediated entry. Indeed, replacing these regions with the respective HLA-DM α2 sequences (chimera 6a) ( Fig 2C) resulted in surface expression (Fig 2E, S2 Data) and viral entry similar to that of wild-type HLA-DR (Figs 2F and S3B, S2 Data). In fact, cell surface expression patterns of all the chimeras were unaffected when we replaced any other individual motif (loops 4 to 7, β-sheets 3 to 7) with the corresponding sequence of HLA-DM ( Fig 2E, S2 Data). Yet, only chimeras 6e to 6g supported viral entry, with infection rates of 40% to 70% relative to HLA-DR ( Fig 2F, S2 Data), suggesting that loop 5, β-sheet 6, and loop 6 are either not essential for the interaction with H18 or contain critical residues that are also conserved in the corresponding HLA-DM sequences. Chimeras 6b, 6c, 6d, 6h, and 6j failed to mediate VSV-H18 infection (Figs 2F and S3B, S2 Data), suggesting that β-sheet 3, loop 4, β-sheet 4, β-sheet 6, loop 7, and β-sheet 7 of the HLA-DR α2 domain are supporting the interaction with H18. These regions form 2 structural motifs on the surface of HLA-DR, which we refer to as surface 1 (S1, made of β-sheet 3, loop 4, and β-sheet 4) and surface 2 (S2, made of β-sheet 6, loop 7, and β-sheet 7) (Fig 2G). The 19 residues of S1 and S2 combined account for 73% to 86% of the α2 MHC-II residues predicted by the models to interact with H18 (Figs 2H and S4D).

Conserved amino acids in loop 4 and β-sheet 6 of the HLA-DR α2 domain are critical for H18-mediated entry
The putative H18-binding site in S1 consists of β-sheet 3, loop 4, and β-sheet 4, with loop 4 forming a prominent kink (Fig 2G, left panel). We hypothesized that this unusual structural feature might contribute to its interaction with H18, and thus we sought to identify the residues within loop 4 required for virus entry. We performed targeted mutagenesis of loop 4 to render a version of HLA-DR/DM-α2 chimera 6c, which would be permissive to H18-mediated infection. While loop 4 of classical MHC-II molecules comprises 9 amino acids, loop 4 of HLA-DM exhibits a deletion of T154 ( Fig 3A). We therefore inserted T154 into chimera 6c, designated chimera 6c1, and determined its efficient cell surface expression (Fig 3B, S3 Data), showing the H18 binding surfaces S1 and S2. β-sheet 3, loop 4, and β-sheet 4 (S1) are in violet, and β-sheet 6, loop 7, and β-sheet 7 (S2) in pink. (H) Surface representation of the HLA-DR structure highlighting S1 and S2 among the residues that constitute the H18 binding sites predicted by model 1. The bar graph highlights the percentage of these residues (black coloring) among the predicted H18 interacting residues. For statistical analysis, one-way ANOVA followed by Dunnett test was performed. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant. HLA-DR, human leukocyte antigen DR; MHC-II, major histocompatibility complex class II.
https://doi.org/10.1371/journal.pbio.3002182.g002 Error bars represent SD. For statistical analysis, one-way ANOVA followed by Dunnett test was performed for panels while infection with VSV-H18 failed (Figs 3C and S3C, S3 Data). Starting from chimera 6c1, we then replaced amino acids H149, S150, and F157 of HLA-DM with the HLA-DR residues N149, G150, and V157 to obtain chimera 6c2. This construct was expressed at the cell surface ( Fig 3B, S3 Data) and allowed efficient virus entry (Figs 3C and S3C, S3 Data). Subsequent backward mutagenesis of the individual residues N149H, G150S, and V157F, in the chimera 6c2 context, indicated that N149H alone (chimera 6c4) was sufficient to prevent H18-mediated entry (Figs 3C and S3C, S3 Data) revealing a crucial function of N149. In line with this finding, evolutionary conservation analysis using the ConSurf Database [21,22] showed a striking cross-species conservation of this position based on 300 sequence homologs (Conservation score: 9/9) ( Fig  3A, S3 Data). To further validate the central role of N149 for H18-mediated entry, we introduced the same HLA-DM-derived substitution N149H into the wild-type HLA-DR (HLA-DR N149H ). HLA-DR N149H exhibited a slightly lower cell surface expression ( Fig 3D, S3 Data), and an infection rate decreased to 35% relative to the wild-type HLA-DR ( Fig 3E and 3F, S3 Data). Since chimera 6c5, carrying the S150G substitution, supported minimal virus entry (10%) (Fig 3C), we speculated that G150 was also involved in virus entry to some extent. Indeed, a double substitution of N149H and G150S in wild-type HLA-DR (HLA-DR N149H+G150S ) resulted in an VSV-H18 infection rate �5% (Fig 3E and 3F) despite its efficient surface expression ( Fig 3D). To confirm the critical role of N149 and G150 for viral entry, we tested the ability of HLA-DR N149H+G150S to promote cell fusion with H18-expressing cells. While wild-type HLA-DR supported cell fusion with H18-expressing cells and supported polykaryon formation, HLA-DR N149H+G150S failed to mediate cell fusion under the same conditions.
To investigate whether the decreased ability of HLA-DR N149H and HLA-DR N149H+G150S to mediate viral entry is not caused by an overall disruption of the MHC-II structure, we tested their function in a T cell activation assay (Fig 3H). For this purpose, we coexpressed the wildtype HLA-DRA, HLA-DRA N149H , or HLA-DRA N149H+G150S with the β chain encoded by the HLA-DRB1 allele 01 (genotype: HLA-DRB1*01:01, serotype: HLA-DR1) in BHK21 cells (S5A- S5D Fig, S5 Data). The MHC-II-expressing BHK21 cells were then loaded with a peptide (HA 307-319 ) and cocultured with the CH7C17 Jurkat T cell line expressing the transgenic TCR HA1.7, which specifically recognizes HA 307-319 loaded on HLA-DR1 ( Fig 3H) [23]. As shown in Figs 3I and S5D and S3 and S5 Data files, wild-type HLA-DR and both variants efficiently activated T cells in a peptide-dependent manner as judged by the expression of the T cell activation marker CD69, confirming that neither N149H nor N149H+G150S adversely affected the overall structural integrity of the HLA-DR complex. These results show that the highly conserved amino acids N149 and (to a lesser extent) G150 in loop 4 of HLA-DR α2 are critical for mediating H18-mediated cell entry.
The architecture of the pocket formed by β-sheet 6, loop 7, and β-sheet 7 of HLA-DR α2 domain is crucial for H18-mediated entry The second putative H18-binding surface (S2) consists of β-sheet 6, loop 7, and β-sheet 7 ( Fig  2G). S2 forms a shallow pocket in HLA-DR and a protrusion in HLA-DM (Fig 4A and 4B), prompting us to speculate that the architecture of this region is important for virus entry. The HLA-DR pocket comprises 2 highly conserved hydrophobic amino acids buried at its base: V190 in β-sheet 6 (conservation score: 9/9) and L195 in loop 7 (conservation score: 8/9) (Figs 4C and S4E, S4 Data) To test the importance of structural integrity of the S2 pocket for viral entry, we B-E, and unpaired Student t test was used for panel I. * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant. EV, empty vector; HLA-DR, human leukocyte antigen DR; TCR, T cell receptor.
To demonstrate the importance of the amino acids N149, G150, and V190 in HLA-DR for authentic H18N11 entry, we infected HEK293T cells transiently expressing mutant HLA-DR V190A and HLA-DR N149H+G150S . Indeed, H18N11 entry was barely detectable in cells expressing these mutant HLA-DR complexes (Fig 4I, S4 Data), confirming the importance of the identified residues for cell entry in context of authentic H18N11. Likewise, VSV-H17 entry was greatly diminished in cells expressing HLA-DR V190A and HLA-DR N149H+G150S , suggesting that H17 and H18 utilize similar key residues for cell entry (Fig 4J, S4 Data).

N149, G150, and V190 are also critical for H18-mediated infection in context of MHC-II of the Jamaican fruit bat
We next sought to determine whether residues N149, G150, and V190, which we identified as critical for H18-mediated entry in the context of human HLA-DR, are similarly relevant in the HLA-DR homolog of the Jamaican fruit bat (Aj-DR), a natural reservoir species of H18N11 [24]. Following transient reconstitution, wild-type Aj-DR complex was well expressed at the cell surface of HEK293T cells (Fig 4K, S4 Data) and supported infection of VSV-H18 (Fig 4L,  S4 Data). In comparison, Aj-DR harboring the double substitution N149H+G150S (Aj-DR N149H+G150S ) or the single V190A (Aj-DR V190A ) mutation reduced the infection rate of VSV-H18 by 80% and 65%, respectively, compared with wild-type Aj-DR (Fig 4L). This suggests that highly conserved amino acids within the MHC-II α2 subunit are required for H18-mediated infection in different species.

Conserved amino acids in α1 and β1 domains of HLA-DR modulate H18-mediated entry
As shown in Fig 1F, both the α1 and the β1 domains of HLA-DR have a role in H18-mediated entry. Since these domains contribute to the formation of the peptide binding groove almost equally, we tested whether the presence and/or identity of a high-affinity peptide would affect VSV-H18 infection. To this end, we expressed HLA-DR1 molecules having either the HA 307-319 peptide or the CLIP 87-101 peptide covalently fused to the N-terminus of the beta chain (HA 307-319 -HLA-DR1 and CLIP 87-101 -HLA-DR1; S6A Fig). Whereas the correct positioning of the fused peptide into the binding groove for HA 307-319 -HLA-DR1 was confirmed by a T cell activation assay (S6B Fig, S6 Data), this was not possible for CLIP 87-101 -HLA-DR1 due to the restriction of the CH7C17 Jurkat T cells to the HA 307-319 peptide. Both CLIP 87-101 and HA 307-319 peptides slightly increased surface expression but substantially improved viral entry (S6C and S6D Fig, S6 Data). We speculate that covalent fusion of a high-affinity peptide stabilizes the MHC-II molecule and thereby facilitates viral entry in a sequence-independent manner. Indeed, binding of a peptide to an empty MHC-II was shown to increase stability at both neutral and acidic pH [25].
To further determine the importance of highly conserved amino acids within the α1 and the β1 domains of HLA-DR for viral entry, individual amino acids therein were substituted with their HLA-DM counterparts or alanine when identical (S7A Fig). Only surface-exposed amino acids that were present in 75% of all MHC-II homologs and also had a conservation score >8 (ConSurf-DB Score) were considered. All the corresponding MHC-II mutants were surface expressed at levels comparable to wild-type HLA-DR (S7B Fig, S7 Data). Viral entry of VSV-H18 was only substantially impaired (>30% reduction of entry positive cells) in cells expressing the MHC-II mutants M61F (α1 domain) or N91A (β1 domain) (S7C Fig, S7 Data). Importantly, M61 of the α1 domain forms part of the predicted H18-binding site, which further supports our in silico modeling approach (S7D Fig, S1 Table).

Discussion
We elucidated the central role of the α2 domain of the MHC-II molecule in H18-mediated cell entry by generating sets of chimeras between the classical human MHC-II, HLA-DR, and the nonclassical MHC-II molecule, HLA-DM. Within the α2 domain, we identified the highly conserved amino acid residues N149 and V190 as critical for both H18-and H17-mediated infection. Single substitutions of these amino acids in HLA-DR prevented viral entry but did not affect the structural integrity of the HLA-DR molecules as these mutants were still able to accommodate antigenic peptides and activate T cells. The fact that these HLA-DR mutants were able to activate T cells demonstrates their overall structural integrity and clustering ability. The latter, which is critical for T cell activation, may also provide the avidity required for virus attachment and entry [19,26,27]. The α2 and β2 domains of MHC-II molecules are C1-set Ig-like domains [28], which makes them well suited to act as platforms for interactions with other proteins [29]. Indeed, the α2 domain, which we found to be crucial for H18N11 entry, was previously shown to bind TIRC7, a negative regulator of T cell activity [30]. Furthermore, the HLA-DR:CD4 and HLA-DR:HLA-DM interactions consist of large multidomain interfaces in which the α2 domain is a major contributor [31][32][33]. Our in silico structural modeling suggests that the interaction with H18 occurs through an interface involving the α2 domain and parts of the α1 and β1 domains in HLA-DR. Despite the involvement of multiple domains, we hypothesize that the H18:HLA-DR interaction is of relatively low affinity as classical biochemical approaches have yet failed to detect direct binding [11]. However, according to our predicted model, 1 H18 homotrimer can bind to 3 MHC-II complexes (S4F and S4G  Fig), which would allow clustering of MHC-II at the viral entry site and provide the avidity required for host cell binding and subsequent uptake. Similar clustering of entry factors is also required for the uptake of classical IAV due to the low affinity of individual HA-sialic acid interactions [34,35].
On a genetic level, the MHC-II α-and β-chains are markedly conserved among all mammalian species with the obvious exception of the polymorphic residues that mainly cluster in the β1 domain [16]. Usage of such a conserved receptor allows bat IAV to infect a wide range of New World bat species, including the phylogenetically only distantly related Neotropical fruit bats, Artibeus spp. (family Phyllostomidae), and Velvety free-tailed bat, Molossus molossus (family Molossidae) [8]. However, despite the ability to utilize MHC-II of diverse mammalian species and the wide geographical distribution of seropositive bats across Central and South America, there is as of yet no evidence for natural infection of non-bat species with bat IAVs. This might suggest that there are additional molecular and/or ecological hurdles, which so far have prevented a spill over to other mammals including humans.

MHC-II expression plasmids
cDNA sequences encoding V5-or HA-tagged wild-type, and chimeric MHC-II α-and βchains were synthesized (Genewiz) and cloned into the pCAGGS vector via NotI and XhoI restriction enzymes (S1A

MHC-II surface expression
HEK293T cells seeded to approximately 70% confluency in 24-well plates were transfected with 250 ng each of the respective MHC-II α-and β-chain using Lipofectamine 2000 (Thermo Fisher, Germany). The next day, cells were detached and washed by pipetting up and down gently with FACS buffer (PBS supplemented with 2% FCS). After centrifugation at 1,200 rpm for 5 min at 4˚C, cells were stained primarily with anti-V5 rabbit antibody (Abcam, catalog no. ab9116, 1:500) and anti-HA mouse antibody (Sigma Aldrich, catalog no. H3663, 1:500) for 30 min on ice. Following another washing and centrifugation step, cells were secondarily stained with BV421 goat anti-rabbit antibody (BD Biosciences, catalog no. 565014, 1:200) and APC goat anti-mouse antibody (BD Biosciences, catalog no. 550826, 1:200) for 30 min on ice. Zombie NIR Fixable Viability Kit (BioLegend, catalog no. 423105, 1:1000) was used to assess live versus dead status of cells. After a final wash and centrifugation step, cells were resuspended in FACS buffer, transferred to a FACS tube, and surface expression of MHC-II heterodimer was analyzed with a BD FACS Canto II (BD Biosciences) flow cytometer.

Virus infections
HEK293T cells seeded to approximately 70% confluency in 24-well plates were transfected with 250 ng each of the respective MHC-II α-and β-chain using Lipofectamine 2000 (Thermo Fisher, Germany). For VSV-H18 infection, cells were infected 24 h posttransfection at an MOI of 0.05 in infection medium [11]. At 24 h postinfection, cells were detached, washed, centrifuged, and stained as described for MHC-II surface expression. After staining and washing, cells were fixed in 2% PFA in PBS for 20 min on ice, washed, and centrifuged at 1,500 rpm for 10 min at 4˚C. After a final wash and centrifugation step, cells were resuspended in FACS buffer, transferred to a FACS tube, and MHCII surface expression as well as the frequency of infected GFP-positive cells were analyzed with a BD FACS Canto II (BD Biosciences) or BD LSRFortessa (BD Biosciences) flow cytometer.
For H18N11 infection, cells were infected 24 h posttransfection at an MOI of 5 in infection medium supplemented with 0.2 μg/ml TPCK trypsin. At 24 h postinfection, cells were resuspended in infection medium, washed, and centrifuged as described for MHC-II surface expression. Subsequently, cells were primarily stained with a rabbit polyclonal anti-H18 serum (1:100) [11] for 30 min on ice. After washing in FACS buffer and centrifugation at 1,200 rpm for 5 min at 4˚C, cells were stained with BV421 goat anti-rabbit antibody (BD Biosciences, catalog no. 565014, 1:200) and Alexa Fluor 488-conjugated HA tag monoclonal antibody (Thermo Fisher, catalog no. A-21287, 1:200) for 30 min on ice. After washing and centrifugation, cells were fixed in 2% PFA in PBS for 20 min on ice, washed, and centrifuged at 1,500 rpm for 10 min at 4˚C. After a final wash step, cells were resuspended in FACS buffer, transferred to a FACS tube, and MHC-II surface expression as well as the frequency of H18N11-infected Alexa Fluor 488-positive cells were analyzed with BD LSRFortessa (BD Biosciences) flow cytometer.
Fluorescent images of GFP-positive VSV-H18-infected cells were acquired on a Zeiss Observer.ZI inverted epifluorescence microscope (Carl Zeiss) equipped with an AxioCamMR3 camera using a 10× objective.

Molecular docking
The HDOCK server (http://hdock.phys.hust.edu.cn/) [36][37][38] was used to computationally construct the three-dimensional (3D) complex model of H18:MHC-II using crystal structures of MHC-II molecules and H18 HA deposited in the PDB and its default hybrid docking protocol. Focusing on the HA1 region that binds the sialic acid receptor of conventional IAVs [39,40] and acquired amino acid mutations that increase replication competence of H18N11 [24], the PDB entry 4K3X [8], (chains A, C, E) of H18 were designated as the "interactor" and MHCII (PDB ID 1DLH, chains A, B) [41] as the "ligand." After a global sampling of putative binding orientations at 15˚rotational intervals, HDOCK provided docking results, including 10 top models based on its scoring function. Among these top 10 docking models, the top 2 models with the lowest docking score (below −200; the most possible binding model) and confidence score greater than 0.7 (very likely to bind), in addition to the criteria of using the HA head domain, not engaging the HLA-DR peptide binding groove, and being in upright orientation, were selected. Pymol was used for 3D structure visualization of H18:MHC-II complex model and PISA was used to calculate buried surface areas.

Conservation analysis
To determine the cross-species conservation of amino acids within the α-and β-chain of HLA-DR, we performed a ConSurf-DB analysis [21,22] for 1DLH chain A and B (PDB DOI: 10.2210/pdb1DLH/pdb) [41]. The calculation was conducted on 300 hits out of 1,872 (αchain) and 5,558 (β-chain) homologs, which were CT-HIT unique at 95% threshold. Where necessary, resulting conservation scores were plotted for our regions of interest using Graph-Pad Prism.
Supporting information S1 Table.  Loops and β-sheets are both numbered 1 to 7. (D) Surface representation of HLA-DR, highlighting S1 and S2 within the H18 binding site predicted by model 2 (left), and a bar graph depicting this in percentages, with S1 and S2 residues highlighted in black (right). (E) Surface representation of the crystal structure of HLA-DR (PDB code:1DLH) [41], highlighting the H18 binding surfaces (S1 and S2) in the α2 domain of MHC-II (left) and stick representation of the residues of S2 highlighting the highly conserved basic amino acids at the base of the pocket (right).